Tatsuru Arai

β-Estradiol protects hippocampal CA1 neurons against transient forebrain ischemia in gerbil

Beta-estradiol has been considered to be a neurotrophic agent, but its in vivo effect on gerbils with transient forebrain ischemia has not yet been demonstrated. In the first set of the present experiments, we infused beta-estradiol at a dose of 0.05 or 0.25 microg/day for 7 days into the lateral ventricles of normothermic gerbils starting 2 h before 3-min forebrain ischemia. Beta-estradiol infusion at a dose of 0.25 microg/day prevented significantly the ischemia-induced reduction of response latency time as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the hippocampal CA1 region as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with beta-estradiol than in those receiving saline infusion. Beta-estradiol at a dose of 1.25 microg/day was ineffective and occasionally increased the mortality of experimental animals. Since the total brain content of exogenous beta-estradiol at 12 h after forebrain ischemia was estimated to be less than 145 ng, the second set of experiments focused on the neurotrophic action of beta-estradiol at concentrations around 100 ng/ml in vitro. Beta-estradiol at concentrations of 1-100 ng/ml facilitated the survival and process extension of cultured hippocampal neurons, but it did not exhibit any significant radical-scavenging effects at the concentration range. On the other hand, 100 microg/ml of beta-estradiol, even though failing to support hippocampal neurons in vitro, effectively scavenged free radicals in subsequent in vitro studies, as demonstrated elsewhere. These findings suggest that beta-estradiol at a dose of 0.25 microg/day prevents ischemia-induced learning disability and neuronal loss at early stages after transient forebrain ischemia, possibly via a receptor-mediated pathway without attenuating free radical neurotoxicity.

Increase of glutamate uptake in astrocytes: A possible mechanism of action of volatile anesthetics

Background: Glutamate is the most ubiquitous excitatory neurotransmitter in the vertebrate central nervous system. Astrocytes play an important role in terminating glutamatergic neurotransmission by removing released glutamate from the synaptic cleft. The authors examined the effects of several anesthetics on the glutamate uptake activity of astrocytes. Methods: Cultured astrocytes from hippocampi of rat embryos were incubated with solution containing [sup 3 H]glutamate, which was pre‐equilibrated with 0–4% halothane at 37 degrees Celsius. The uptake activity was evaluated as the amount of radioactivity per cell of protein. Results: When the reaction solution was equilibrated with 4% halothane, glutamate uptake increased to about 165% of the control. The effect of halothane was dose‐dependent, and a significant augmentation (30–50%) of glutamate uptake was observed at a range in clinical use concentrations (1–2%). On the other hand, the uptake of gamma‐aminobutyric acid, an inhibitory transmitter, was hardly affected by 1–4% halothane. The effect of halothane on glutamate uptake was also examined in neuron‐rich culture, and similar augmentation was observed, although the extent was less than that in astrocyte culture. Biochemical subcellular fractions (i.e., glial plasmalemmal vesicles and synaptosomes) were also examined, however, only slight (not significant) increase was detected in the glutamate uptake activity. Other volatile anesthetics, such as enflurane, isoflurane, and sevoflurane, also enhanced glutamate uptake, whereas the intravenous anesthetics ketamine and pentobarbital showed no effect on glutamate uptake. Conclusions: The increase of glutamate uptake by astrocytes in the presence of volatile anesthetics potentially attenuates excitatory synaptic transmission in the entire central nervous system, a finding that may explain in part the action of volatile anesthetics.

The effects of 17β-estradiol on ischemia-induced neuronal damage in the gerbil hippocampus

The effects of 17beta-estradiol, a potent estrogen, on ischemia-induced neuronal damage, membrane depolarization and changes in intracellular Ca2+ concentration were studied in gerbil hippocampi. The histological outcome evaluated seven days after 3 min of transient forebrain ischemia in hippocampal CA1 pyramidal cells was improved by high doses of 17beta-estradiol (30 microg, i.c.v. and 4 mg/kg, i.p.), whereas low doses of 17beta-estradiol (3 and 10 microg, i.c.v.) showed no protective effect. Administration of 17beta-estradiol did not affect the changes in the direct current potential shift in ischemia in the hippocampal CA1 area at any dosage. A hypoxia-induced intracellular Ca2+ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices. Pretreatment of 17beta-estradiol (4 mg/kg, injected i.p. 1 h before decapitation) suppressed the increase in the intracellular concentration of Ca2+ due to the in vitro hypoxia, affecting both the onset of the increase and the extent. The in vitro hypoxia in the Ca2+-free condition induced an elevation of the intracellular concentration of Ca2+, although the increase was gradual. Pretreatment of 17beta-estradiol (4 mg/kg, i.p.) also inhibited this elevation. These findings imply that high doses of 17beta-estradiol protect the neurons from ischemia by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, as well as by inhibiting the influx of Ca2+ from the extracellular space.

The effect of hypothermia on H2O2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus

The changes in the extracellular concentration of hydrogen peroxide (H2O2) in gerbil hippocampus during ischemia and reperfusion were investigated by microdialysis coupled with fluorometry of dichlorofluorescin oxidation. In a normothermic condition (37.5 degrees C), a transient forebrain ischemia for 5 or 10 min produced a significant increase in hippocampal H2O2 immediately after the start of ischemia. The duration of this elevation after reperfusion was significantly shorter in gerbils subjected to 5 min of ischemia than in those subjected to 10 min of ischemia. Hypothermia at both 34 degrees C and 30 degrees C inhibited the increase in the H2O2 concentration during ischemia and reperfusion in gerbils subjected to 5 min of ischemia. In gerbils subjected to 10 min of ischemia, hypothermia delayed the onset of the increase in the H2O2 concentration and shortened the duration of the elevated H2O2 concentration. Hypothermia improved the histological outcome in the hippocampal CA1 neurons 7 days after ischemia. These findings suggest that the suppression of H2O2 production in ischemia and reperfusion is a possible mechanism of brain protection by hypothermia.

Origin of Ischemia‐Induced Glutamate Efflux in the CA1 Field of the Gerbil Hippocampus: An In Vivo Brain Microdialysis Study

Abstract: In vivo brain microdialysis experiments were performed in the gerbil to evaluate the origin of accumulation of extracellular glutamate under transient ischemia. Microdialysis probes were positioned in the CA1 field of the hippocampus in which proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals had been induced by 5‐min ischemia 10–14 days before the microdialysis experiment; in the white matter of the cerebral cortex, which contained few neurons, few presynaptic terminals, and many astrocytes; or in the histologically normal CA1 field of the hippocampus, and then 5‐ or 20‐min ischemia was induced. When 5‐min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter of the cerebral cortex, whereas a significant increase in glutamate (15‐fold) was observed in the histologically normal CA1 field. When 20‐min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter during the first 10 min after the onset of 20‐min ischemia, but remarkable ischemia‐induced increases in glutamate were observed during the last 10 min of 20‐min ischemia in both areas. An excessive increase in glutamate (100‐fold) was observed during 20‐min ischemia in the normal CA1 field of the hippocampus. When a probe was positioned in the CA1 field of the hippocampus in which presynaptic terminals of Schaffer collaterals and commissural fibers had been eliminated by bilateral kainate injections into the lateral ventricles 4–7 days before the microdialysis experiment and then 5‐min ischemia was induced, a significant increase in glutamate was observed during the last half of 5‐min ischemia. These results suggest that the efflux of glutamate from astrocytes does not contribute to the large ischemia‐induced glutamate accumulation in the CA1 field of the hippocampus during 5‐min ischemia but contributes to the ischemia‐induced increase in glutamate level during ischemia with a longer duration and that ischemia‐induced efflux of glutamate in the CA1 field during 5‐min ischemia originates mainly from neuronal elements: presynaptic terminals and postsynaptic neurons.

Dexamethasone Aggravates Ischemia-Induced Neuronal Damage by Facilitating the Onset of Anoxic Depolarization and the Increase in the Intracellular Ca2+ Concentration in Gerbil Hippocampus

The Ca2+ mobilization across the neuronal membrane is regarded as a crucial factor in the development of neuronal damage in ischemia. Because glucocorticoids have been reported to aggravate ischemic neuronal injury, the effects of dexamethasone on ischemia-induced membrane depolarization, histologic outcome, and changes in the intracellular Ca2+ concentration in the gerbil hippocampus were examined in vivo and in vitro. The effects of metyrapone, an inhibitor of glucocorticoid synthesis, were also evaluated. Changes in the direct-current potential shift in the hippocampal CA1 area produced by transient forebrain ischemia for 2.5 minutes were compared among animals pretreated with dexamethasone (3 μg, intracerebroventricularly), metyrapone (100 mg/kg, intraperitoneally), and saline. The histologic outcome was evaluated 7 days after ischemia by assessing the delayed neuronal death in the hippocampal CA1 pyramidal cells of these animals. A hypoxia-induced intracellular Ca2+ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effect of dexamethasone (120 μg/L in the medium) on the cytosolic Ca2+ accumulation was examined. The effect in a Ca2+-free ischemialike condition was also investigated. Preischemic administration of dexamethasone reduced the onset latency of ischemia-induced membrane depolarization by 22%, and aggravated neuronal damage in vivo. In contrast, pretreatment with metyrapone improved the histologic outcome. The onset time of the increase in the intracellular concentration of Ca2+ provoked by in vitro hypoxia was advanced in dexamethasone-treated slices. The Ca2+-free in vitro hypoxia reduced the elevation compared with that in the Ca2+-containing condition. Treatment with dexamethasone facilitated the increase on both the initiation and the extent in the Ca2+-free condition. Aggravation of ischemic neuronal injury by endogenous or exogenous glucocorticoids is thus thought to be caused by the advanced onset times of both the ischemia-induced direct-current potential shift and the increase in the intracellular Ca2+ concentration.

Measurement of the extracellular H2O2 in the brain by microdialysis

This paper reports on the protocol for the determination of H2O2 in the brain using in vivo microdialysis coupled with fluorometry of dichlorofluorescin oxidation. We applied this protocol to monitor changes in the concentration of H2O2 in the brain, in vivo, during ischemia and reperfusion. Using this method, changes in the level of H2O2 in the brain during ischemia and reperfusion were effectively determined. The present protocol provides a novel tool to study the production of reactive oxygen species in the brain.

Delayed Neuronal Death is Induced without Postischemic Hyperexcitability: Continuous Multiple-Unit Recording from Ischemic CA1 Neurons

It has been proposed that neuronal hyperexcitability during postischemic chronic stage mediates delayed neuronal death in the hippocampal CA1 region. In the present study, multiple-unit spike discharges were continuously recorded from hippocampal CA1 neurons of the awake Mongolian gerbil for 5 days after 5 min of ischemia. Before ischemia, CA1 neurons showed burst-like spike discharges (so-called complex spikes). Spike discharges disappeared 8–40 s after the onset of 5-min ischemia and reappeared 5–30 min after recirculation. The frequency of discharges gradually increased but did not return to the preischemic level. The amplitude of the spike discharges was smaller than that recorded before ischemia and the number of spikes composing the burst-like discharges diminished. CA1 neurons did not show any hyperexcitability for 5 days. However, histological examinations revealed widespread neuronal death in the CA1 region. These results indicate that the delayed neuronal death in the hippocampal CA1 region is induced without postischemic neuronal hyperexcitability.

Lidocaine protects hippocampal neurons against ischemic damage by preventing increase of extracellular excitatory amino acids : a microdialysis study in Mongolian gerbils

Extracellular levels of amino acids in the hippocampal CA1 region of the gerbil were analyzed by a microdialysis-HPLC procedure. Transient forebrain ischemia produced significant increase in aspartate, glutamate, glycine and taurine (760%, 1070%, 190% and 1210%, respectively), and neuronal blockade by perfusion with a lidocaine (4 mM)-containing medium resulted in 67%, 79%, 58% and 59% reduction in the peak values of each amino acid, respectively. On the other hand, an intracerebroventricular administration of lidocaine, 0.8 mumol or more, produced a protective effect against delayed damage of hippocampal CA1 pyramidal cells, which was caused by bilateral carotid artery occlusion for 4 min. The results suggest that lidocaine may protect neurons against ischemic damage, by preventing the ischemia-induced rise of extracellular concentration of excitatory amino acid.

Dexamethasone Changes Brain Monoamine Metabolism and Aggravates Ischemic Neuronal Damage in Rats

BACKGROUND Glucocorticoids have been reported to aggravate ischemic brain damage. Because changes in the activities of various neuronal systems are closely related to the outcome of ischemic damage, the authors evaluated the effects of dexamethasone on the monoaminergic systems and ischemic neuronal damage. METHODS The right middle cerebral artery was occluded for 2 h, and the tissue concentrations of monoamines and their metabolites were determined in the cerebral cortex and the striatum of rats. The turnover of 5-hydroxytryptamine was compared in animals injected with saline and those injected with dexamethasone twice (2 mg/kg in each injection) by evaluating the probenecid-induced accumulation of 5-hydroxyindoleacetic acid. The turnovers of norepinephrine and dopamine were estimated from the alpha-methyl-p-tyrosine-induced depletion of norepinephrine and dopamine, respectively. The effect of dexamethasone on the infarct volume was evaluated by triphenyltetrazolium chloride stain in rats subjected to 2 h of occlusion. RESULTS Dexamethasone did not affect the cortical 5-hydroxytryptamine or 5-hydroxyindoleacetic acid contents. However, it suppressed the turnover of the cortical 5-hydroxytryptamine on both sides. Dexamethasone reduced the turnover of the striatal 5-hydroxytryptamine and facilitated the dopamine turnover. In rats subjected to 2 h of occlusion and 2 h of reperfusion, the infarct volume was 10.5 times greater in the group that received dexamethasone than in the animals that received saline. CONCLUSIONS Dexamethasone suppresses the inhibitory serotonergic system and facilitates the excitatory dopaminergic system in the rat telencephalon. This may be a mechanism by which dexamethasone aggravates ischemic neuronal injury.